Means and methods for treating cancer

ABSTRACT

The present invention relates to a pharmaceutical composition comprising a compound of formula (I) 
     
       
         
         
             
             
         
       
     
     and methods of treating or preventing cell proliferation disorders comprising administering to a subject a therapeutically active amount or a preventive amount of such a compound.

The present invention relates to a pharmaceutical composition comprisinga compound of formula (I)

wherein R¹ is selected from halogen, substituted or unsubstituted C₁ toC₆ alkyl, substituted or unsubstituted C₁ to C₆ alkenyl, substituted orunsubstituted C₁ to C₆ alkinyl, substituted or unsubstituted C₁ to C₆acyl, C₁ to C₆ haloalkyl, H, NO₂, OH, SH, NH₂, C₁ to C₆ alkoxy, CN andN(CH₃)₂, substituents being selected from OH, SH and NH₂, the term“substituted” providing for 1 or 2 substituents; R² is selected from H,substituted or unsubstituted C₁ to C₆ alkyl, substituted orunsubstituted C₁ to C₆ alkenyl and substituted or unsubstituted C₁ to C₆alkinyl, substituents being selected from OH, SH and NH₂, the term“substituted” providing for 1 or 2 substituents; A, B, C and D areindependently selected from N and CH; Z is selected from NH, S, CH₂, adirect single bond, and a direct double bond; V is selected fromsubstituted or unsubstituted heterocycloalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, heteroatoms in said heterocycloalkyl andsaid heteroaryl being selected from N, O and S, the total number ofheteroatoms in said heterocycloalkyl and said heteroaryl being 1, 2, 3or 4, the term “substituted” providing for 1, 2 or 3 substituents,substituents being selected from C₁ to C₆ alkyl, C₁ to C₆ alkenyl, C₁ toC₆ alkinyl, C₁ to C₆ acyl, C₁ to C₆ haloalkyl, NO₂, OH, SH, NH₂, C₁ toC₆ alkoxy, CN and N(CH₃)₂; and m is 0 or an integer selected from 1, 2,3, 4 and 5.

In this specification, a number of documents including patentapplications and manufacturer's manuals are cited. The disclosure ofthese documents, while not considered relevant for the patentability ofthis invention, is herewith incorporated by reference in its entirety.More specifically, all reference documents are incorporated by referenceto the same extent as if each individual document was specifically andindividually indicated to be incorporated by reference.

Isocitrate dehydrogenase, also known as IDH, is an enzyme whichparticipates in the citric acid cycle. It catalyzes the third step ofthe cycle: the oxidative decarboxylation of isocitrate, producingalpha-ketoglutarate (α-ketoglutarate or α-KG) and CO₂ while convertingNAD+ to NADH. This is a two-step process, which involves oxidation ofisocitrate (a secondary alcohol) to oxalosuccinate (a ketone), followedby the decarboxylation of the carboxyl group beta to the ketone, formingalpha-ketoglutarate. Another isoform of the enzyme catalyzes the samereaction; however this reaction is unrelated to the citric acid cycle,is carried out in the cytosol as well as the mitochondrion andperoxisome, and uses NADP+ as a cofactor instead of NAD+.

Mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 were originallyidentified in glioma and acute myeloid leukemia (AML) patients (Mardis,E. R., Ding, L., Dooling, D. J., Larson, D. E. McLellan, M. D., Chen,K., Koboldt, D. C., Fulton, R. S., Delehaunty, K. D., McGrath, S. D., etal. (2009), Recurring mutations found by sequencing an acute myeloidleukemia genome. N Engl J Med 361, 1058-1066; Parsons, D. W., Jones, S.,Zhang, X., Lin, J. C., Leary, R. J., Angenendt, P., Mankoo, P., Carter,H., Siu, I. M., Gallia, G. L., et al. (2008), An integrated genomicanalysis of human glioblastoma multiforme. Science 321, 1807-1812; andYan, H., Parsons, D. W., Jin, G., McLendon, R., Rasheed, B. A., Yuan,W., Kos, I., Batinic-Haberle, I., Jones, S., Riggins, G. J., et al.(2009), IDH1 and IDH2 mutations in gliomas. N Engl J Med 360, 765-773)),but are increasingly found in a diverse set of other tumor entities likechondrosarcoma (Amary, M. F., Bacsi, K., Maggiani, F., Damato, S.,Halai, D., Berisha, F., Pollock, R., O'Donnell, P., Grigoriadis, A.,Diss, T., et al. (2011), IDH1 and IDH2 mutations are frequent events incentral chondrosarcoma and central and periosteal chondromas but not inother mesenchymal tumours. J Pathol 224, 334-343), lymphoma (Cairns, R.A., Iqbal, J., Lemonnier, F., Kucuk, C., de Leval, L., Jais, J. P.,Parrens, M., Martin, A., Xerri, L., Brousset, P., et al. (2012), IDH2mutations are frequent in angioimmunoblastic T-cell lymphoma. Blood 119,1901-1903)), melanoma (Shibata, T., Kokubu, A., Miyamoto, M., Sasajima,Y., and Yamazaki, N. (2011), Mutant IDH1 confers an in vivo growth in amelanoma cell line with BRAF mutation. Am J Pathol 178, 1395-1402), andthyroid cancer (Murugan, A. K., Bojdani, E., and Xing, M. (2010),Identification and functional characterization of isocitratedehydrogenase 1 (IDH1) mutations in thyroid cancer. Biochem Biophys ResCommun 393, 555-559). IDH mutations were identified in 15-30% of AML andother myeloid malignancies, including myelodysplastic syndromes (MDS)and myeloproliferative neoplasms (MPN) (Paschka, P., Schlenk, R. F.,Gaidzik, V. I., Habdank, M., Kronke, J., Bulllinger, L., Spath, D.,Kayser, S., Zucknick, M., Gotze, K., et al. (2010), IDH1 and IDH2mutations are frequent genetic alterations in acute myeloid leukemia andconfer adverse prognosis in cytogenetically normal acute myeloidleukemia with NPM1 mutation without FLT3 internal tandem duplication. JClin Oncol 28, 3636-3643; Thol, F., Damm, F., Wagner, K., Gohring, G.,Schlegelberger, B., Hoelzer, d., Lubbert, M., Heit, W., Kanz, L.,Schlimok, G., et al. (2010), Prognostic impact of IDH2 mutations incytogenetically normal acute myeloid leukemia, Blood 116, 614-616; andWagner, K., Damm, F., Gohring, G., Gorlich, K., Heuser, M., Schafer, I.,Ottmann, O., Lubbert, M., Heit, W., Kanz, L., et al. (2010), Impact ofIDH1 R132 mutations and an IDH1 single nucleotide polymorphism incytogenetically normal acute myeloid leukemia: SNP rs11554137 is anadverse prognostic factor. J Clin Oncol 28, 2356-2364). IDH mutationsreside in the active site of the enzyme and participate in isocitratebinding. In many instances, they are missense alterations affectingarginine-140 (R140) residue in the IDH2 protein. IDH1 mutants lack thewild-type enzyme's ability to convert isocitrate to a α-ketoglutaratebut gains a neomorphic activity which leads to the conversion of α-KG tothe oncometabolite 2-hydroxyglutarate (2HG) (Dang, L., White, D. W.,Gross, S., Bennett, B. D., Bittinger, M. A., Driggers, E. M., Fantin, V.R., Jang, H. G., Jin, S., Keenan, M. C., et al. (2009),Cancer-associated IDH1 mutations produce 2-hydroxyglutarate, Nature 462,739-744; Gross, S., Cairns, R. A., Minden, M. D., Driggers, E. M.,Bittinger, M. A., Jang, H. G., Sasaki, M., Jin, S., Schenkein, D. P.,Su, S. M., et al. (2010), Cancer-associated metabolite2-hydroxyglutarate accumulates in acute myelogenous leukemia withisocitrate dehydrogenase 1 and 2 mutations, J Exp Med 207, 339-344; andWard, P. S., Cross, J. R., Lu, C., Weigert, O., Abel-Wahab, O., Levine,R. L., Weinstock, D. M., Sharp, K. A., and Thompson, C. B. (2012),Identification of additional IDH mutations associated withoncometabolite R(−)-2-hydroxyglutarate production. Oncogene 31,2491-2498). It has been discovered that the product of neoactivity, 2HG,can be significantly elevated in cancer cells harbouring IDH1/2mutations.

Given the disease relevance of isocitrate dehydrogenase, in particularmutated forms thereof, there is an unmet need of compounds targetingisocitrated dehydrogenase, preferably mutated forms thereof. Relatedthereto, there is an ongoing need for means and methods of treatingcancer.

This technical problem is solved by the subject-matter of the enclosedclaims.

Accordingly in the first aspect, the present invention relates to apharmaceutical composition comprising a compound of formula (I)

wherein R¹ is selected from halogen, substituted or unsubstituted C₁ toC₆ alkyl, substituted or unsubstituted C₁ to C₆ alkenyl, substituted orunsubstituted C₁ to C₆ alkinyl, substituted or unsubstituted C₁ to C₆acyl, C₁ to C₆ haloalkyl, H, NO₂, OH, SH, NH₂, C₁ to C₆ alkoxy, CN andN(CH₃)₂, substituents being selected from OH, SH and NH₂, the term“substituted” providing for 1 or 2 substituents; R² is selected from H,substituted or unsubstituted C₁ to C₆ alkyl, substituted orunsubstituted C₁ to C₆ alkenyl and substituted or unsubstituted C₁ to C₆alkinyl, substituents being selected from OH, SH and NH₂, substituentsbeing selected from OH, SH and NH₂, the term “substituted” providing for1 or 2 substituents; A, B, C and D are independently selected from N andCH; Z is selected from NH, S, CH₂, a direct single bond, and a directdouble bond; V is selected from substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted cycloalkyl, substitutedor unsubstituted aryl, substituted or unsubstituted heteroaryl,heteroatoms in said heterocycloalkyl and said heteroaryl being selectedfrom N, O and S, the total number of heteroatoms in saidheterocycloalkyl and said heteroaryl being 1, 2, 3 or 4, the term“substituted” providing for 1, 2 or 3 substituents, substituents beingselected from C₁ to C₆ alkyl, C₁ to C₆ alkenyl, C₁ to C₆ alkinyl, C₁ toC₆ acyl, C₁ to C₆ haloalkyl, NO₂, OH, SH, NH₂, C₁ to C₆ alkoxy, CN andN(CH₃)₂; and m is 0 or an integer selected from 1, 2, 3, 4 and 5.

It is noted that for each of moieties R₁, R₂ and V, the term“substituted” has a different meaning, which different meaning is clearfrom the wording of the embodiment of the first aspect. E.g.,substituents selected from C₁ to C₆ alkyl, C₁ to C₆ alkenyl, C₁ to C₆alkinyl, C₁ to C₆ acyl, C₁ to C₆ haloalkyl, NO₂, OH, SH, NH₂, C₁ to C₆alkoxy, CN and N(CH₃)₂ are specific for substituted forms of moiety Vsuch as substituted heterocycloalkyl.

The compounds according to the present invention, owing to the presenceof a positive charge on the nitrogen atom depicted in formula (I), areto be provided with a counterion. Suitable counterions can be chosen bythe skilled person without further ado. Preferred counterions includethe halogenides, in particular Cl⁻ and Br⁻; as well as acetate andsalicylate.

The skilled person can synthesize the compounds according to the presentinvention without further ado. The key step is the synthesis of3-substituted pyrrolidine rings which can be performed as described, forexample, in Laborde, Tetrahedron Lett. 33, 6607-6610 (1992) or Kurkin etal., Chem. Heterocycl. Comp. 43, 34-40 (2007). A corresponding generalscheme is shown below.

Moiety R in this scheme has its counterpart in the moiety comprising Vand bound to N in formula (I).

Preferably, said haloalkyl is CF₃. Preferably, said alkoxy is OCH₃.

By virtue of their capability to bind and inhibit one or more mutantand/or wild-type isoforms of isocitrate dehydrogenase, the compoundsaccording to the present invention are useful in the treatment ofcancer, preferably those forms of cancer which are known to beisocitrate dehydrogenase-dependent.

The pharmaceutical composition may comprise or consist of one or morecompounds in accordance with the present invention. In case it comprisesone or more compounds according to the present invention, furtherconstituents may be present. These further constituents may bepharmaceutically active compounds. Any further pharmaceutically activeagents, to the extent they are present, may be selected from cancer,chemotherapeutic agents, antibodies directed against targets involved incancer and hormones. Also, therapeutic treatment in accordance with thepresent invention may be combined with surgical treatment, in particularsurgical removal of tumors. In other words, the subject or patient to betreated with the pharmaceutical composition of the invention may be asubject or patient who has undergone, is undergoing, or will undergosuch surgical treatment.

Having said that, it is preferred that one or more compounds inaccordance with the present invention are the only pharmaceuticallyactive compound(s) comprised in said pharmaceutical composition.

To the extent said pharmaceutical composition comprises one or morecompounds according to the present invention, it is furthermoreenvisaged that carriers, excipients and/or fillers as known in thepharmaceutical art are present.

The pharmaceutical composition will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient, the site of delivery of thepharmaceutical composition, the method of administration, the schedulingof administration, and other factors known to practitioners. Theeffective amount of the pharmaceutical composition for purposes hereinis thus determined by such considerations.

The skilled person knows that the effective amount of pharmaceuticalcomposition administered to an individual will, inter alia, depend onthe nature of the compound. For example, the total pharmaceuticallyeffective amount of pharmaceutical composition administered parenterallyor orally per dose may be in the range of about 1 μg compound /kg/day to50 mg compound /kg/day of patient body weight, although, as noted above,this will be subject to therapeutic discretion. More preferably, thisdose is at least 0.01 mg compound /kg/day, and most preferably forhumans between about 0.01 and about 50 mg compound /kg/day. If givencontinuously, the pharmaceutical composition is typically administeredat a dose rate of about 1 μg/kg/hour to about 500 μg/kg/hour, either by1-4 injections per day or by continuous subcutaneous infusions, forexample, using a mini-pump. An intravenous bag solution may also beemployed. Alternatively, the compound may be administered orally in oneor up to 5 doses per day at a dose range of about 1 μg compound /kg/dayto about 50 mg compound /kg/day of patient body weight. The length oftreatment needed to observe changes and the interval following treatmentfor responses to occur appears to vary depending on the desired effect.The particular amounts may be determined by conventional tests which arewell known to the person skilled in the art.

Pharmaceutical compositions of the invention may be administered orally,rectally, parenterally including intravenously, intrathecally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or transdermal patch), bucally, or as an oralor nasal spray.

Pharmaceutical compositions of the invention preferably comprise apharmaceutically acceptable carrier. By “pharmaceutically acceptablecarrier” is meant a non-toxic solid, semisolid or liquid filler,diluent, encapsulating material or formulation auxiliary of any type.The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

The pharmaceutical composition is also suitably administered bysustained release systems. Suitable examples of sustained-releasecompositions include semi-permeable polymer matrices in the form ofshaped articles, e.g., films, or mirocapsules. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. etal., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate)(R. Langer et al., J. Biomed. Mater. Res. 15:167-277 (1981), and R.Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langeret al., Id.) or poly-D-(−)-3-hydroxybutyric acid (EP 133,988). Sustainedrelease pharmaceutical composition also include liposomally entrappedcompound. Liposomes containing the pharmaceutical composition areprepared by methods known per se: DE 3,218,121; Epstein et al., Proc.Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl.Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046;EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos.4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes areof the small (about 200-800 Angstroms) unilamellar type in which thelipid content is greater than about 30 mol. percent cholesterol, theselected proportion being adjusted for the optimal therapy.

For parenteral administration, the pharmaceutical composition isformulated generally by mixing it at the desired degree of purity, in aunit dosage injectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation.

Generally, the formulations are prepared by contacting the components ofthe pharmaceutical composition uniformly and intimately with liquidcarriers or finely divided solid carriers or both. Then, if necessary,the product is shaped into the desired formulation. Preferably thecarrier is a parenteral carrier, more preferably a solution that isisotonic with the blood of the recipient. Examples of such carriervehicles include water, saline, Ringer's solution, and dextrosesolution. Non aqueous vehicles such as fixed oils and ethyl oleate arealso useful herein, as well as liposomes. The carrier suitably containsminor amounts of additives such as substances that enhance isotonicityand chemical stability. Such materials are non-toxic to recipients atthe dosages and concentrations employed, and include buffers such asphosphate, citrate, succinate, acetic acid, and other organic acids ortheir salts; antioxidants such as ascorbic acid; low molecular weight(less than about ten residues) peptides, e.g., polyarginine ortripeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids, such as glycine, glutamic acid, aspartic acid, or arginine;monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, glucose, manose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;counterions such as sodium; and/or nonionic surfactants such aspolysorbates, poloxamers, or PEG.

The components of the pharmaceutical composition to be used fortherapeutic administration must be sterile. Sterility is readilyaccomplished by filtration through sterile filtration membranes (e.g.,0.2 micron membranes). Therapeutic components of the pharmaceuticalcomposition generally are placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The components of the pharmaceutical composition ordinarily will bestored in unit or multi-dose containers, for example, sealed ampoules orvials, as an aqueous solution or as a lyophilized formulation forreconstitution. As an example of a lyophilized formulation, 10-m1 vialsare filled with 5 ml of sterile-filtered 1% (w/v) aqueous solution, andthe resulting mixture is lyophilized. The solution for infusion orinjection is prepared by reconstituting the lyophilized compound(s)using bacteriostatic Water-for-Injection. The compounds described hereincan, for example, be administered per orally, by injection,intravenously, intraarterially, subdermally, intraperitoneally,intramuscularly, or subcutaneously.

Generally speaking, as regards the number of carbon atoms in any moietydefined to be C₁ to C₆, it is preferred that the moiety contains between1 and 4 carbon atoms. Particularly preferred is C₁ and C₂.

In a preferred embodiment, A, B, C and D are CH. Also preferred is thateither A or B is N, the remainder of A, B, C and D being CH.

In a further preferred embodiment, Z is CH₂ or NH. Particularlypreferred is that Z is CH₂.

In a further preferred embodiment, m is 0, 1 or 2, preferably 1.

In a further preferred embodiment, V is substituted or unsubstitutedheterocycloalkyl, preferably substituted or unsubstituted piperazinyl,more preferably substituted or unsubstituted piperazin-2-yl, and yetmore preferably 1,4-dimethyl-piperazin-2-yl.

In other words, preferred pharmaceutical compositions are thosecomprising a compound of formula (Ia)

wherein R¹ is selected from halogen, substituted or unsubstituted C₁ toC₆ alkyl, substituted or unsubstituted C₁ to C₆ alkenyl, substituted orunsubstituted C₁ to C₆ alkinyl, substituted or unsubstituted C₁ to C₆acyl, C₁ to C₆ haloalkyl, H, NO₂, OH, SH, NH₂, C₁ to C₆ alkoxy, CN andN(CH₃)₂, substituents being selected from OH, SH and NH₂, the term“substituted” providing for 1 or 2 substituents; R² is selected from H,substituted or unsubstituted C₁ to C₆ alkyl, substituted orunsubstituted C₁ to C₆ alkenyl and substituted or unsubstituted C₁ to C₆alkinyl, substituents being selected from OH, SH and NH₂, substituentsbeing selected from OH, SH and NH₂, the term “substituted” providing for1 or 2 substituents; A, B, C and D are independently selected from N andCH; Z is selected from NH, S, CH₂, a direct single bond, and a directdouble bond; V is selected from substituted or unsubstitutedheterocycloalkyl, heteroatoms in said heterocycloalkyl being selectedfrom N, O and S, the total number of heteroatoms in saidheterocycloalkyl being 1, 2, 3 or 4, the term “substituted” providingfor 1, 2 or 3 substituents, substituents being selected from C₁ to C₆alkyl, C₁ to C₆ alkenyl, C₁ to C₆ alkinyl, C₁ to C₆ acyl, C₁ to C₆haloalkyl, NO₂, OH, SH, NH₂, C₁ to C₆ alkoxy, CN and N(CH₃)₂; and m is 0or an integer selected from 1, 2, 3, 4 and 5.

A particularly preferred class of compounds in accordance with thepresent invention are the compounds of formula (V)

R¹ and R² are as defined in accordance with the first aspect orpreferably in accordance with the preferred embodiments described below.

In a further preferred embodiment, R¹ is halogen, preferably F, Cl or Br

In a further preferred embodiment, R² is H.

In a further preferred embodiment of the first aspect of the inventionor any of its above described preferred embodiments, the stereochemistryof said compound is as shown in formula (II)

In particularly preferred embodiments of the pharmaceutical compositionof the present invention, said compound is a compound of formula (III)

This compound is also referred to as HMS-101 herein.

The compound of this invention may be modified by appending appropriatefunctionalities to enhance selected biological properties, e.g.,targeting to a particular tissue. Such modifications are known in theart and include those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

Preferably, the compounds according to the present invention arefurthermore characterized, more preferably inherently characterized, bythe following features. In particular, it is preferred said compound (a)inhibits a mutant form of at least one isoform of isocitratedehydrogenase, said mutant preferably being characterized by one or moreof the following: (i) reduced formation of α-ketoglutarate by theencoded isocitrate dehydrogenase as compared to the wild-type isocitratedehydrogenase; (ii) increased formation of 2-hydroxyglutarate by theencoded isocitrate dehydrogenase as compared to the wild-type isocitratedehydrogenase; (iii) no increased formation of 2-hydroxyglutarate by theencoded isocitrate dehydrogenase as compared to the wild-type isocitratedehydrogenase; (iv) aberrant splicing such as absence of exon 7 in thespliced mRNA; (v) a missense mutation affecting R132 in human isocitratedehydrogenase 1; (vi) a missense mutation affecting R172 in humanisocitrate dehydrogenase 2; (vii) a missense mutation affecting R140 inhuman isocitrate dehydrogenase 2; (viii) a missense mutation affectingR100 in human isocitrate dehydrogenase 1; and (ix) a missense mutationaffecting G97 in human isocitrate dehydrogenase 1; and/or (b) binds to apocket in the three-dimensional structure of said mutant form of atleast one isoform of isocitrate dehydrogenase, said pocket being absentin the corresponding wild-type form(s).

The term “isoform of isocitrate dehydrogenase” includes isocitratedehydrogenase 1 and isocitrate dehydrogenase 2. The sequences of humanisocitrate dehydrogenase 1 and human isocitrate dehydrogenase 2 areherewith included as SEQ ID NOs: 1 and 2.

The specific mutations according to items (v) to (ix) are preferredmutations which lead to the increased formation of 2-hydroxyglutarate.Preferred missense mutations affecting R132 in accordance with aboveitem (v) are R132H (also referred to as IDHmut herein) as well as R132C.In particular these two mutants have been further investigated in theExamples enclosed herewith.

It is understood that item (a) provides for the presence of one or moreof features (i) to (ix). To give an example, aberrant splicing inaccordance with (a)(iv) may be observed in a mutant form of isocitratedehydrogenase which differs from the corresponding wild-type only inthat said aberrant splicing occurs, or, in the alternative, aberrantsplicing may affect a mutant of isocitrate dehydrogenase in which casethere would be concomitant presence of aberrant splicing and a furthermutation, said further mutation not necessarily being related to theprocess of splicing.

As regards sub-items (ii) and (iii), we note that preference is given tothose mutants which are characterized by the increased formation of2-hydroxyglutarate, in particular of R-2-hydroxyglutarate (also known asD-2-hydroxyglutarate). As noted above, 2-hydroxyglutarate is beingviewed as a oncometabolite and accordingly as a hallmark of IDHmutant-associated cancer. More specifically, and as noted above, it isR-2-hydroxyglutarate which is a marker for IDH mutant-associated cancer,in particular leukemia. Elevated levels of R-2-hydroxyglutarate may bemeasured in terms of the ratioR-2-hydroxyglutarate/S-2-hydroxyglutarate. Exemplary data can be foundin FIGS. 2 and 6 enclosed herewith.

Yet, the present inventors surprisingly observed that mutated isocitratedehydrogenase, in particular an alternatively spliced isoform of mutatedIDH1, may promote leukemogenesis independently of 2-hydroxyglutarate. Asa consequence, it is preferred that compounds according to the presentinvention also target mutant forms of isocitrate dehydrogenase which arenot characterized by increased formation of 2-hydroxyglutarate.

Furthermore, the inventors surprisingly observed that compounds inaccordance with the present invention were effective also in certainleukemia patients which have wild-type isocitrate dehydrogenase. As aconsequence, while isocitrate dehydrogenase is still viewed to be a keymolecule for the neoplasia seen in these patients, it is important tonote that also targeting the wild-type form of isocitrate dehydrogenasemay be beneficial. This applies in particular, but not only, toindividuals or patients with increased expression and/or activity of atleast one isoform of isocitrate dehydrogenase.

The term “increased expression” refers to an expression at the mRNA orprotein level which is elevated as compared to the average expression atthe mRNA or protein level, respectively, in healthy individuals.Similarly, the term “increased activity” refers to increased activity ascompared to the average activity in healthy individuals. The term“activity” preferably is the enzymatic activity, in particular theformation of alpha-ketoglutarate or 2-hydroxyglutarate. The term“increased” refers in this context to an increase of 200, 300, 400, 500,600, 700, 800, 900 or 1000% or more percent. Also envisaged is atwofold, threefold, fourfold, fivefold to tenfold or higher increase ofexpression or activity.

In a preferred embodiment, said compound binds to and/or inhibits saidmutant form to a higher degree than (to) the corresponding wild-typeform.

As stated above, such type of specificity is generally preferred. On theother hand, it is not a strict necessity, noting that also patientshaving cancer and a wild-type isocitrate dehydrogenase benefit fromtreatment with compounds in accordance with the present invention.

In view of the documented relevance of isocitrate dehydrogenase forcancer in general and various specific forms thereof, the presentinvention provides in a second aspect a compound as defined above foruse in a method of treating or preventing a cell proliferation disorder,said disorder preferably being a hyperplasia, tumor or cancer, morepreferably being selected from leukemia including myeloid leukemia suchas acute myeloid leukemia (AML), myeloid malignancies includingmyelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN),glioma, lymphoma, and solid tumors including prostate cancer, thyroidcancer, sarcomas such as fibrosarcoma and chondrosarcoma, and melanoma.

In a preferred embodiment, the subject suffering from or being at riskof said disorder is (a) characterized by increased expression and/oractivity of at least one isoform of isocitrate dehydrogenase; or (b)heterozygous or homozygous with regard to a mutant form of at least oneisoform of isocitrate dehydrogenase, said mutant preferably beingcharacterized by one or more of the following: (i) reduced formation ofα-ketoglutarate by the encoded isocitrate dehydrogenase as compared tothe wild-type isocitrate dehydrogenase; (ii) increased formation of2-hydroxyglutarate by the encoded isocitrate dehydrogenase as comparedto the wild-type isocitrate dehydrogenase; (iii) no increased formationof 2-hydroxyglutarate by the encoded isocitrate dehydrogenase ascompared to the wild-type isocitrate dehydrogenase; (iv) aberrantsplicing such as absence of exon 7 in the spliced mRNA; (v) a missensemutation affecting R132 in human isocitrate dehydrogenase 1; (vi) amissense mutation affecting R172 in human isocitrate dehydrogenase 2;(vii) a missense mutation affecting R140 in human isocitratedehydrogenase 2; (viii) a missense mutation affecting R100 in humanisocitrate dehydrogenase 1; and (ix) a missense mutation affecting G97in human isocitrate dehydrogenase 1.

In a third aspect, the present invention provides a method of treatingor preventing a cell proliferation disorder, said disorder preferablybeing a hyperplasia, tumor or cancer, more preferably being selectedfrom leukemia including myeloid leukemia such as acute myeloid leukemia(AML), myeloid malignancies including myelodysplastic syndrome (MDS) andmyeloproliferative neoplasms (MPN), glioma, lymphoma, and solid tumorsincluding prostate cancer, thyroid cancer, sarcomas such as fibrosarcomaand chondrosarcoma, and melanoma, said method comprising the step ofadministering a therapeutically active amount or a preventive amount ofa compound as defined above.

In a further aspect, the present invention provides a mouse beingcharacterized by (a) an activated gene of the HOXA cluster, preferablyHOXA 9; and (b) (ba) increased expression and/or activity of at leastone isoform of isocitrate dehydrogenase; or (bb) presence of a mutantform of at least one isoform of isocitrate dehydrogenase, said mutantpreferably being characterized by one or more of the following: (i)reduced formation of α-ketoglutarate by the encoded isocitratedehydrogenase as compared to the wild-type isocitrate dehydrogenase;(ii) increased formation of 2-hydroxyglutarate by the encoded isocitratedehydrogenase as compared to the wild-type isocitrate dehydrogenase;(iii) no increased formation of 2-hydroxyglutarate by the encodedisocitrate dehydrogenase as compared to the wild-type isocitratedehydrogenase; (iv) aberrant splicing such as absence of exon 7 in thespliced mRNA; (v) a missense mutation affecting R132 in human isocitratedehydrogenase 1; (vi) a missense mutation affecting R172 in humanisocitrate dehydrogenase 2; (vii) a missense mutation affecting R140 inhuman isocitrate dehydrogenase 2; (viii) a missense mutation affectingR100 in human isocitrate dehydrogenase 1; and (ix) a missense mutationaffecting G97 in human isocitrate dehydrogenase 1.

Preferred means of activating a gene of the HOXA cluster, preferablyHOXA 9, are retroviral overexpression, lentiviral overexpression, oroverexpression in a transgenic model. It is understood thatoverexpression is a preferred implementation of activation. Bothactivation and overexpression are defined by reference to the parentstrain from which the mouse according to the present invention has beenderived from. A preferred parent strain is strain C57BL6. Preferred isan increase in expression as compared to the parent strain of at least10%, at least 20%, at least 30%, at least 40%, at least 50% or more.Also envisaged is twofold, threefold, fourfold, fivefold, tenfold orhigher overexpression.

The above defined mouse model is a preferred model for in vivo screensdirected to the identification and/or validation of compounds targetingisocitrate dehydrogenase in a disease-relevant context. Morespecifically, said mouse model recapitulates leukemogenesis. The presentinventors used said mouse model in order to establish that production of2-hydroxyglutarate, while being a preferred feature characterizingaberrantly proliferating cells with mutated isocitrate dehydrogenase, isnot a requirement for oncogenic activity of mutated isocitratedehydrogenase.

The figures show:

FIG. 1: IC50 values for IDH1wt and IDH1mut treated with compound HMS-101for 72 hours. The compound described herein has an IC50 of 1 μM onIDH1mut cancer cells in vitro, whereas it has a 12 times higher IC50 (12μM) on IDH1wt cells in vitro as analysed by Alamar blue cell viabilityassay.

FIG. 2: D-2HG levels from IDH1mut cells treated with compound HMS-101(drug) or solvent control. The compound described herein has reduced theneomorphic activity of the IDH1mutant enzyme, i.e. the production of2HG, as evident by dramatically reduced intracellular 2HG levels inIDH1mutant cells treated with compound HMS-101 for 72 hours at 10 μMdose of compound as compared to the control treated cells.

FIG. 3: Colony forming assay of primary human AML cells with and withoutIDH1 mutation treated with solvent control (DMSO) or compound HMS-101.Human CD34⁺ hematopoietic stem and progenitor cells from healthy donorswere used as controls. The compound HMS-101 reduced the colony formingpotential of primary human AML cells harboring the IDH1 mutation whencompared to primary human AML cells with wildtype IDH1 and cells fromhealthy donors.

FIG. 4: Rate of apoptosis of primary human AML cells with and withoutIDH1 mutation treated with solvent control (DMSO) or compound HMS-101.Human CD34⁺ hematopoietic stem and progenitor cells from healthy donorswere used as controls. The compound HMS-101 increased the rate ofapoptosis of primary human AML cells harboring the IDH1 mutationsignificantly more compared to primary human AML cells with wildtypeIDH1 and cells from healthy donors.

FIG. 5: Survival of mice treated with HMS-101.

FIG. 6: Ratio of R-2-hydroxyglutarate (R-2HG) to S-2-hydroxyglutarate(S-2HG) in serum from mice treated with HMS-101 or solvent control (PBS)at 9 weeks after treatment. Serum from 3 mice were pooled per indicatedmeasurement (9 mice for PBS, 12 mice for HMS-101).

The following examples illustrate the invention.

EXAMPLE 1 Affinity of Compound with IDH Mutant

The predicted affinity (in silico modeling) of compound HMS-101 to IDH1is comparable to IDH inhibitor compounds published recently¹⁵ (ΔG≈8kcal/mol). The compound HMS-101 has significantly better predictedaffinity (ΔΔG≈10 kcal/mol) for IDHmut (R132H) in the presence of NADPthan to wildtype IDH1. In the absence of NADP, the compound describedherein has selectivity towards mutant but not as pronounced as withpresence of NADP. This high selectivity is favorable compared tocompounds recently patented¹⁶, which show selectivity to the IDH1 mutantby ΔΔG≈1-2 kcal/mol.

EXAMPLE 2 Toxicity Testing in Animals

The compound described herein has been given to mice at a dose of 1 mgintraperitoneally daily for two weeks. The compound was well toleratedby the animals with no visible signs of toxicity.

EXAMPLE 3 In Vitro Proliferation of Cancer Cells (IC50)

The compound described herein has an IC50 of 1 μM on IDH1mut cancercells in vitro, whereas it has a 12 times higher IC50 (12 μM) on IDH1 wtcells in vitro as analysed by Alamar blue cell viability assay (FIG. 1).

EXAMPLE 4 Neoactivity of IDHmut Enzyme

The compound described herein has reduced the neomorphic activity of theIDH1 mutant enzyme, i.e. the production of 2HG, as evident bydramatically reduced intracellular 2HG levels in IDH1mutant cellstreated with compound HMS-101 for 72 hours at 10 μM dose of compound ascompared to the control treated cells (FIG. 2).

EXAMPLE 5 Effect of Compound HMS-101 on the Colony Forming Potential ofPrimary Human Acute Myeloid Leukemia Cells

The compound HMS-101 reduced the colony forming potential of primaryhuman AML cells harboring the IDH1 mutation when compared to primaryhuman AML cells with wildtype IDH1 and cells from healthy donors (FIG.3).

EXAMPLE 6 Effect of Compound HMS-101 on the Rate of Apoptosis of PrimaryHuman AML Cells

The compound HMS-101 increased the rate of apoptosis of primary humanAML cells harboring the IDH1 mutation significantly more compared toprimary human AML cells with wildtype IDH1 and cells from healthy donors(FIG. 4).

EXAMPLE 7 In Vivo Effect of HMS-101

HoxA9 immortalized bone marrow cells from C57BL/6J mice were transducedwith retroviral vectors expressing IDH1R132C and GFP. One millionGFP-expressing sorted cells were injected intravenously inlethally-irradiated syngeneic recipient mice, accompanied by alife-sparing dose of 1×10⁵ freshly isolated bone marrow cells fromsyngeneic mice. One cohort of mice was treated with HMS-101 at a dose of1 mg/mouse for 5 days/week starting 5 days after transplantation. Thecontrol cohort was treated with PBS for 5 days/week starting 5 daysafter transplantation. The solid line represents the survival ofPBS-treated mice, the dotted line represents the survival ofHMS-101-treated mice. Follow-up and treatment are ongoing. As allcontrol mice are dead, the graph indicates a survival benefit forHMS-101 treated mice.

EXAMPLE 8 2-Hydroxyglutarate as Marker for Leukemia

2-hydroxyglutarate (R-2HG), in particular elevated values thereof,typically measured in terms of the ratio between R- and S-form(R-2HG/S-2HG) is a marker for leukemia in accordance with the presentinvention. As shown in FIG. 6, treatment with a preferred compound inaccordance with the present invention (HMS-101) causes a decrease of theratio R-2HG/S-2HG in treated mice. This establishes both the usefulnessof the mentioned ratio as a marker for leukemia and the beneficialeffects of HMS-101.

1. A pharmaceutical composition comprising a compound of formula (I)

wherein R¹ is selected from halogen, substituted or unsubstituted C₁ toC₆ alkyl, substituted or unsubstituted C₁ to C₆ alkenyl, substituted orunsubstituted C₁ to C₆ alkynyl, substituted or unsubstituted C₁ to C₆acyl, C₁ to C₆ haloalkyl, H, NO₂, OH, SH, NH₂, C₁ to C₆ alkoxy, CN andN(CH₃)₂, substituents being selected from OH, SH and NH₂, the term“substituted” providing for 1 or 2 substituents; R² is selected from H,substituted or unsubstituted C₁ to C₆ alkyl, substituted orunsubstituted C₁ to C₆ alkenyl and substituted or unsubstituted C₁ to C₆alkynyl, substituents being selected from OH, SH and NH₂, the term“substituted” providing for 1 or 2 substituents; A, B, C and D areindependently selected from N and CH; Z is selected from NH, S, CH₂, adirect single bond, and a direct double bond; V is selected fromsubstituted or unsubstituted heterocycloalkyl, substituted orunsubstituted cycloalkyl, substituted or unsubstituted aryl, substitutedor unsubstituted heteroaryl, heteroatoms in said heterocycloalkyl andsaid heteroaryl being selected from N, O and S, the total number ofheteroatoms in said heterocycloalkyl and said heteroaryl being 1, 2, 3or 4, the term “substituted” providing for 1, 2 or 3 substituents,substituents being selected from C₁ to C₆ alkyl, C₁ to C₆ alkenyl, C₁ toC₆ alkenyl, C₁ to C₆ acyl, C₁ to C₆ haloalkyl, NO₂, OH, SH, NH₂, C₁ toC₆ alkoxy, CN and N(CH₃)₂; and m is 0 or an integer selected from 1, 2,3, 4 and
 5. 2. The pharmaceutical composition of claim 1, wherein A, B,C and D are CH.
 3. The pharmaceutical composition of claim 1, wherein Zis CH₂ or NH.
 4. The pharmaceutical composition of claim 1, wherein m is0, 1 or
 2. 5. The pharmaceutical composition of claim 1, wherein V issubstituted or unsubstituted heterocycloalkyl.
 6. The pharmaceuticalcomposition of claim 1, wherein R¹ is halogen.
 7. The pharmaceuticalcomposition of claim 1, wherein R² is H.
 8. The pharmaceutical ordiagnostic composition of claim 1, wherein the stereochemistry of saidcompound is as shown in formula (II)


9. The pharmaceutical composition of claim 7, wherein said compound is acompound of formula (III)


10. The pharmaceutical composition of claim 9, wherein said compound isa compound of formula (IV)

11-12. (canceled)
 13. A method of treating or preventing a cellproliferation disorder, said method comprising the step of administeringto a subject suffering from or being at risk of the cell proliferationdisorder a therapeutically active amount or a preventive amount of acompound as defined in claim
 1. 14. A mouse being characterized by (a)an activated gene of the HOXA cluster, preferably HOXA 9; and (b) (ba)increased expression and/or activity of at least one isoform ofisocitrate dehydrogenase; or (bb) presence of a mutant form of at leastone isoform of isocitrate dehydrogenase, said mutant preferably beingcharacterized by one or more of the following: (i) reduced formation ofα-ketoglutarate by the encoded isocitrate dehydrogenase as compared tothe wild-type isocitrate dehydrogenase; (ii) increased formation of2-hydroxyglutarate by the encoded isocitrate dehydrogenase as comparedto the wild-type isocitrate dehydrogenase; (iii) no increased formationof 2-hydroxyglutarate by the encoded isocitrate dehydrogenase ascompared to the wild-type isocitrate dehydrogenase; (iv) aberrantsplicing such as absence of exon 7 in the spliced mRNA; (v) a missensemutation affecting R132 in human isocitrate dehydrogenase 1; (vi) amissense mutation affecting R172 in human isocitrate dehydrogenase 2;(vii) a missense mutation affecting R140 in human isocitratedehydrogenase 2; (viii) a missense mutation affecting R100 in humanisocitrate dehydrogenase 1; and (ix) a missense mutation affecting G97in human isocitrate dehydrogenase
 1. 15. The pharmaceutical compositionof claim 4, wherein m is
 1. 16. The pharmaceutical composition of claim5, wherein V is substituted or unsubstituted piperazinyl.
 17. Thepharmaceutical composition of claim 16, wherein V is substituted orunsubstituted piperazin-2-yl.
 18. The pharmaceutical composition ofclaim 17, wherein V is 1,4-dimethyl-piperazin-2-yl.
 19. The method ofclaim 13, wherein the cell proliferation disorder is leukemia, glioma,lymphoma, prostate cancer, thyroid cancer, or sarcoma.
 20. The method ofclaim 19 wherein the subject suffering from or being at risk of saiddisorder is (a) characterized by increased expression and/or activity ofat least one isoform of isocitrate dehydrogenase; or (b) heterozygous orhomozygous with regard to a mutant form of at least one isoform ofisocitrate dehydrogenase.
 21. The method of claim 12, wherein thesubject suffering from or being at risk of said disorder is heterozygousor homozygous with regard to a mutant form of at least one isoform ofisocitrate dehydrogenase and further wherein said mutant form ischaracterized by one or more of the following: (i) reduced formation ofα-ketoglutarate by the encoded isocitrate dehydrogenase as compared tothe wild-type isocitrate dehydrogenase; (ii) increased formation of2-hydroxyglutarate by the encoded isocitrate dehydrogenase as comparedto the wild-type isocitrate dehydrogenase; (iii) no increased formationof 2-hydroxyglutarate by the encoded isocitrate dehydrogenase ascompared to the wild-type isocitrate dehydrogenase; (iv) aberrantsplicing such as absence of exon 7 in the spliced mRNA; (v) a missensemutation affecting R132 in human isocitrate dehydrogenase 1; (vi) amissense mutation affecting R172 in human isocitrate dehydrogenase 2;(vii) a missense mutation affecting R140 in human isocitratedehydrogenase 2; (viii) a missense mutation affecting R100 in humanisocitrate dehydrogenase 1; and (ix) a missense mutation affecting G97in human isocitrate dehydrogenase 1.